This invention relates to the methods of altering the phenotype of birds by introducing foreign DNA into the muscle of birds.
Commercial poultry is an extremely important source of food. However, there has been comparatively little attention given to methods of producing useful changes in the phenotype of birds through genetic engineering techniques. This is unfortunate, because such techniques offer a much more rapid technique for introducing desirable phenotypic traits into birds than classical breeding techniques.
Currently, the most widely investigated method of gene transfection in poultry employs retroviral vectors. Exemplary is Souza et al., J. Exptl. Zool. 232, 465-473 (1984), in which a retroviral vector encoding growth hormone was injected into the vascularized portion of the yolk sac of 9 day old embryos. See also Shuman and Shoffer, Poult. Sci. 65, 1437-1444 (1986); Salter et al., Poultry Sci. 65, 1445-1468 (1986); Salter et al., Virology 157, 236-240 (1987); Bosselman et al., Science 243, 533-535 (1989); and U.S. Pat. No. 5,162,215 to Bosselman et al.
Nabel et al., Science 249 1285-1288 (1990), and Wolff et al., Science 247, 1445-1468 (1990), state that transient expression of 2-5 months may be obtained from direct microinjection of DNA, but do not suggest how these techniques may be applied to genetically engineering poultry. Nabel et al. note that the expression of DNA encoding xcex2-galactosidase injected into porcine arterial segments was limited to the microinjection site. Acsadi et al., New Biologist 3, 71-81 (1991) state that myocardial cells were able to transiently express injected foreign genes.
Simkiss et al., Protoplasma 151, 164-166 (1989) indicate that primordial germ cells of Stage XVII embryos containing endogenous retroviral sequences can be transferred to comparable recipient Stage XVI embryos that lack the retroviral marker by cardiac puncture. At day 17 of incubation, dot blots on recipient birds showed donor DNA to be present in the gonads, and traces of donor DNA to be present in the liver and heart tissues. The expression of the injected DNA molecules was not reported.
PCT patent application Ser. No. US90/01515 discloses a method of delivering a nucleic acid sequence to the interior of a vertebrate cell. Injection of a DNA molecule into poultry was not reported.
In view of the foregoing, an object of the present invention is to provide methods of changing the phenotype of birds through genetic engineering procedures.
An additional object of the present invention is to provide a method of changing the phenotype of birds in which expression of an exogenous DNA sequence is sufficient produce the phenotypic change.
Another object of the present invention is to provide a method of changing the phenotype of birds which is rapid and convenient.
A first aspect of the present invention is a method of altering the phenotype of a bird. The method comprises introducing a DNA molecule into the cells of a bird contained within an egg during in ovo incubation, with the DNA molecule being effective to cause a change in phenotype in the bird after hatch (e.g., a change in growth rate, feed efficiency, disease resistance, or a combination of all of these factors). Introduction of the DNA may be carried out by any suitable means, including injecting the DNA molecule in ovo into any compartment of the egg including the body of the embryo.
Preferably, the egg into which the DNA is introduced is incubated to hatch, and the bird so produced raised to at least an age at which the change in phenotype is expressed.
A second aspect of the present invention is a bird produced by the foregoing methods.
In an illustrative embodiment of the foregoing, the DNA molecule is introduced into muscle tissue of the bird in ovo, preferably by direct microinjection during late embryonic development.
A third aspect of the present invention is a method for altering the phenotype of a bird comprising introducing a DNA molecule into the muscle tissue of a bird contained within an egg during in ovo incubation, wherein the DNA molecule is effective in causing a change in phenotype in the bird after hatch.
A fourth aspect of the present invention is a method for immunizing a bird comprising introducing a DNA molecule into the muscle tissue of a bird contained within in an egg during in ovo incubation, wherein the DNA molecule is effective in inducing an immune response in the bird.
A fifth aspect of the present invention is a method for treating a bird comprising introducing a DNA molecule encoding for an antigen into the muscle tissue of a bird contained within an egg during in ovo incubation in an amount sufficient to neutralize maternal antibodies. In a preferred embodiment, the DNA molecule is introduced at or after the development of immunocompetence by the bird.
A sixth aspect of the present invention is the use of a DNA molecule for the preparation of a medicament for carrying out any of the foregoing methods.
A seventh aspect of the present invention is an apparatus for the introduction of a DNA molecule in an egg during in ovo incubation for carrying out any of the foregoing methods.